The present invention relates generally to the field of biological tissue analysis. More specifically, the present invention relates to a system and method for determining an optimal dilution of a reagent for use in a quantitative immunoassay. There are a variety of different immunoassays for which the claimed invention is applicable including but not limited to tissue-based immunohistochemical, cell-based immunohistochemical analysis such as flow cytometry, and high content screening (HCS) immunohistochemical analysis, enzyme linked immunosorbent assay (ELISA), and western blot assays.
The determination of optimal dilutions of a reagent for a given biological specimen is beneficial to quantitative immunoassays. By way of example, if a reagent is not concentrated enough, then the analysis with the reagent is likely to produce under-detection along with loss of sensitivity at the upper range of the assay. Correspondingly, if the reagent is too concentrated, the reagent is likely to produce over-detection along with loss of sensitivity in the lower range of the assay.
Existing approaches for determining an optimal dilution of a reagent are purely qualitative. The qualitative nature of such an optimization considerably reduces reproducibility of a particular dilution and analysis. Further, such qualitative approach fails to account for a variety of pertinent factors causing reduced reliability of results obtained utilizing such a qualitative optimization.
Immunohistochemistry (IHC) is an immunoassay method for detection of analytes in tissue sections. Traditional IHC assay results have been qualitative in nature, often done by a manual visual assessment through a microscope using a subjective scoring system to indicate a relative amount of analyte present in the tissue sample. In contrast, quantitative IHC analytically measures the amount of one or more analytes of interest in a tissue section. Analytical systems have been developed for quantitative IHC analysis. For example one such system is the AQUA® technology described in U.S. Pat. No. 7,219,016 and in an article entitled “Automated Subcellular Localization and Quantification of Protein Expression in Tissue Microarrays,” Camp et al. 2002 Nature Medicine 8(11)1323-1327. However, even with the introduction of such quantitative analysis for IHC, typically preliminary assays are run to determine optimal concentrations of reagents to be used in the analytical assay and these results are assessed qualitatively, not quantitatively. There is a need for methods for quantitatively determining optimal reagent concentrations for use in quantitative immunoassays, including IHC.